1. Technical Field
The invention relates to protection against Filovirus induced hemorrhagic fevers using recombinant proteins. The invention, more specifically, concerns several recombinant Filovirus structural proteins produced from eukaryotic cells. In the invention, proteins are post-translationally modified and are purified so that native structure is retained, thereby providing material for use as immunogens to elicit protection against virally induced disease.
“Subunit protein” is defined here as any protein derived or expressed independently from the complete organism that it is derived from. Furthermore, a subunit protein may represent a full length native protein sequence or any fraction of the full length native protein sequence. Additionally, a subunit protein may contain in addition to the full length or partial protein sequence, one or more sequences, which may contain sequences that are homologous or heterologous to the organism from which the primary sequence was derived. This definition is significantly broader than the concept of a subunit protein as a single protein molecule that co-assembles with other protein molecules to form a multimeric or oligomeric protein. The subunit proteins of the invention are produced in a cellular production system by means of recombinant DNA methods and, after purification, are formulated in a vaccine.
2. Related Art
Ebola and Marburg viruses are the only two members of the Filovirus family. They are enveloped, negative strand RNA viruses. The viral RNAs have a length of approximately 19 kb and their genome structure is 3′-NP-VP35-VP40-GP-VP30-VP24-L-5′. Upon entry into the cytoplasm of host cells, individual subgenomic, viral mRNA species are transcribed by viral RNA polymerase (RdRp). While the host cell synthesizes viral proteins in the cytoplasm, RNA dependent RNA polymerase (L) in complex with VP35 replicates the complete Filovirus RNA. Viral RNA together with nucleoprotein NP and VP30 forms the nuclear core complex. The nuclear core is inserted into Filovirus envelopes formed by the viral matrix protein VP40 in combination with VP24 and mature viral surface glycoprotein (GP) anchored into the host cell membrane. A schematic diagram of a mature Filovirus particle is shown in FIG. 1.
Marburg virus was first identified in an outbreak that occurred during 1967 in Marburg, Germany. The first human cases were animal handlers, and it was established that the source of infection were monkeys imported from Africa. For the next 9 years there were no reported outbreaks of Filovirus induced hemorrhagic fevers. In 1976, the Zaire subtype of Ebola virus first appeared in an outbreak in the Democratic Republic of Congo (known then as Zaire). A second outbreak occurred in the same year in Sudan caused by the Sudan subtype which showed lower fatality rates. Later two more subtypes were identified, Ebola Ivory-Coast and Ebola Reston. To date a total of 18 Ebola virus outbreaks have caused 1860 cases of human disease and resulted in 1296 deaths. Six Marburg virus outbreaks have to date caused 613 human infections and were fatal in 494 cases (source: WHO). Case fatality rates are dependent on the specific subtype of Filovirus causing the disease and generally vary between 50-90%, with Ebola Zaire and Marburg viruses showing the highest mortality rates. Primates are the only confirmed natural hosts for Filoviruses and human outbreaks can usually be traced back to contact with infected primates. Even though bats could serve as a potential Marburg and Ebola virus reservoir based on observations and laboratory experiments (Swanepoel et al. 1996, Leroy et al. 2005), no natural reservoir has been confirmed to date (Feldmann et al 2004).
Despite relatively few outbreaks, Ebola and Marburg viruses are well known due to their extreme virulence. Both viruses cause fulminant hemorrhagic fevers and death in up to 90% of human infections depending on the infecting strain and route of infection. Although the viruses are endemic only to certain parts of central Africa and the Philippines, the threat of a bioterrorist attack using Ebola virus has raised concern about the virus worldwide. Intentional exposure of non-human primates with aerosolized Ebola virus (Johnson et al. 1995) has demonstrated that a bioterrorist attack using Ebola or Marburg virus is feasible and could affect thousands of people. In outbreaks, it is believed that human-to-human spread only occurs upon direct contact with bodily fluids of an infected patient. However, it has been documented that an unintentional animal to animal infection was possible even without direct body contact of the individually caged rhesus monkeys (Jaax et al. 1995). In addition, recent revelations related to the efforts to produce weaponized Marburg virus in the former Soviet Union (Alibek 1999) reinforced the need to prioritize Ebola/Marburg viruses as a biodefense target. While state of the art medical treatment increases the chances of survival for patients, there are currently no antiviral therapies available to cure the disease, nor a vaccine that protects healthy individuals from infection. Supportive treatment is costly and resource demanding due to the need for strict containment. An Ebola or Marburg virus outbreak, especially in a densely populated urban area, whether caused by natural transmission or terrorist attack, could lead to many casualties and have a dramatic impact on public health systems. A safe and effective vaccine or antiviral treatment that could be used to protect healthcare workers, other at-risk persons, and travelers is therefore highly desirable.
The nucleotide sequence analysis of gene 4 of Marburg virus revealed the basic features of Filovirus surface glycoproteins (singular, “GP”; plural, “GPs”) (Will et al. 1993). Filovirus GPs typically possess a hydrophobic signal peptide at the N-terminus, a hydrophilic external domain, a hydrophobic transmembrane anchor and a small hydrophilic cytoplasmic tail. The signal peptide is removed during the maturation of the glycoproteins and the external domain is heavily glycosylated, with carbohydrates accounting for approximately 50% of the molecular weight of the mature protein. Most research on the biology of Filoviruses focuses on GP, a type I transmembrane glycoprotein. GP is involved in cell entry and is therefore presumed to be a key target for virus neutralization. The Filovirus glycoproteins, on the genomic as well as on the protein level, show strain specific features; Filoviruses can therefore be differentiated and identified based on GP structure. Further analysis of the proteins expressed in Ebola and Marburg virus-infected cells revealed that in addition to the mature GP, Ebola, but not Marburg, viruses express a soluble glycoprotein (sGP). This product results from early translation termination and lacks the majority of the C-terminal region of mature GP (Volchkov et al. 1998). Since it lacks the transmembrane domain it is secreted as dimers into the extracellular space. GP and sGP are synthesized from the same gene. While sGP is synthesized from the direct mRNA transcript, it was shown that the expression of mature GP requires post-transcriptional editing of the mRNA. Expression of the edited GP gene in mammalian cells results in trimerization and the formation of characteristic spikes on the cell surface. In contrast, sGP is secreted as antiparallel-oriented homodimers stabilized by intermolecular disulfide bonds (Volchkova et al. 1998). An Ebola virus mutant was generated from cDNA that included the edited form of the GP gene (expressing mature GP only). This mutant showed a much higher cytotoxicity in comparison to wild-type Ebola virus, which is most likely linked to the much higher concentration of GP present in infected cells (Volchkov et al. 2001). The focus of the current application is on expression of the native mature GP which is expected to form trimeric structures. Immune responses targeting the native structure are more likely to protect individuals from disease than responses targeting non-native (e.g., sGP) structures.
After translation of the post-transcriptionally edited mRNA into the native (non-mutant) polypeptide, preGPer, in the endoplasmatic reticulum, the preGPer polypeptide is heavily glycosylated to form preGP. Furin cleavage separates preGP into two mature subunits, GP1 and GP2, which are linked by an intermolecular disulfide bond (Volchkov et al. 1998). Mutational analysis revealed that inter- and intramolecular disulfide bonding is highly conserved and important for virus entry. Jeffers et al. (2002) suggest that disulfide bonds play a more important role in formation of functional glycoprotein than the presence of N-linked glycosylations. A similar post-translational cleavage into GP1 and GP2 is also observed on Marburg virus GP (Volchkov et al. 2000). A summary of the different pathways of Filovirus GP synthesis can be found in Feldmann et al. 2001.
The surface glycoproteins, i.e., the GPs, may contribute to the cytopathic effects observed in Filovirus infections via a variety of direct and indirect mechanisms. It has been shown that mature Ebola GP alone induces cellular detachment in cell cultures without inducing cell death (Chan et al. 2000). This is in contrast to sGP or GP1 which do not induce cellular detachment. Interestingly, Marburg virus GP does not induce similar detachment. Further characterization of the cytopathicity of GP identified that cell detachment was dependent on dynamin (Sullivan et al., 2005). It has also been shown that native mature GP can trigger activation of primary human macrophages resulting in production of proinflammatory cytokines (Wahl-Jensen et al. 2005). As Filovirus disease is often associated with overproduction of cytokines, this represents a possible mechanism for direct cytopathic effect (see further discussion of pathogenesis of Filovirus disease below). Again, this activity was associated with native, mature GP (trimeric form) and not GP1 and sGP. Furthermore, GP may contribute to the severe pathogenicity of Filovirus infection via indirect mechanisms (e.g., by inhibiting a potent protective immune response to the virus). It has been demonstrated that GP can decrease cell surface expression of MHC class I molecules (Sullivan et al., 2005). This may result in decreased immune responses upon infection. In addition, secretion of sGP as well as shedding of GP1 from virus infected cells (Dolnik et al. 2004) may diminish the activity of neutralizing antibodies.
The Filovirus surface glycoprotein is the only viral protein that is exclusively localized on the surface of the virus particles and possesses structural features to facilitate cell binding and fusion with the cell membrane. It was demonstrated that Ebola GP binds to the cell surface exposed lectins DC-SIGN and DC-SIGNR (Simmons et al. 2003). These lectins are exposed on early infection target cells such as dendritic and other antigen presenting cells and binding of these molecules may facilitate fusion efficiency by changing GP's conformation. Work by Takada et al. (2004) further confirmed the enhancement of Filovirus cell entry caused by galactose- and N-acetylgalactosamine specific C-type lectins (hMGL) present on these early target cells. The role of GP1 in cell surface receptor-binding has been suggested. Extensive mutational analysis has identified the N-terminal third of GP1 as the region most important for this first step of viral entry (Manicassamy et al. 2005). Sequence analysis of GP2 revealed the presence of a putative fusion domain (Volchkov et al. 1992). Ruiz-Arguello et al. (1998) demonstrated in vitro that the putative fusion peptide possesses liposome-binding capability. Mutational analysis using pseudotyped vesicular stomatitis viruses confirmed these findings, suggesting that the fusion process of Ebola virus might mirror that of influenza or human immunodeficiency viruses (Ito et al. 1999). A model was developed based on the hydrophobicity of the fusion peptide and tested in vitro; the testing confirmed that the fusion peptide spans residues 25 to 35 of the GP2 protein (Adam et al. 2004). Gomara et al. (2004) demonstrated further that an internal proline residue in the fusion peptide is responsible for the necessary membrane destabilization to initiate the fusion process. GP2 assembles into a rod-like trimeric structure which resembles that of other viral membrane-fusion proteins such as HIV gp41 or influenza HA2 (Weissenhorn et al. 1998). The crystal structure revealed that an internal GP2 fragment forms a trimer with a central three-stranded coiled coil structure surrounded by shorter C-terminal helices packed in an antiparallel conformation into hydrophobic grooves on the surface of the coiled coils (Malashkevich et al. 1999). Further functional analysis of the coiled-coil interactions revealed their importance in facilitating the entry of Ebola virus into host cells and demonstrated that inhibitors of this interaction could act as efficient antiviral compounds. Thus, in a mature, wild-type GP, the GP2 portion of GP is likely involved in trimerization and fusion, while the GP1 portion is likely involved in binding to the cellular receptors and facilitating entry into target cells.
To date no effective antiviral treatment against Filoviruses is available. Bray (2003) provides a comprehensive review of methods to defend against the use of Filoviruses as biological weapons. In another publication, Geisbert and Jahrling (2004) report the current status of progress towards protection against viral hemorrhagic fevers in general. In addition to immunotherapeutics and antivirals, the publication discusses several potentially supportive interventions targeting the host immune system. Potent antiviral compounds could either target virus proteins or host proteins required for appropriate virus protein processing during the viral lifecycle. The surface glycoprotein GP is a prime antiviral target because of its involvement in host cell entry. DC-SIGN binding can effectively be inhibited with hyperbranched dendritic polymers functionalized with mannose (Rojo and Delgado 2004). Another potential antiviral drug, cyanovirin-N, also exploits the DC-SIGN binding activity of GP and shows activity inhibiting GP-pseudotyped lentivirus entry into HeLa cells (Barrientos et al. 2004c). Another study demonstrated that monomeric as well as dimeric cyanovirin-N show approximately the same antiviral activity (Barrientos et al. 2004b). The importance of endosomal proteolysis by cathepsin B and L for Ebola virus entry was demonstrated by Chandran et al. (2005) and further confirmed by Schornberg et al. (2006). This suggests that cathepsin inhibitors may show activity as anti-Ebola virus drugs.
Filovirus matrix proteins (VP40) possess the general characteristic functionality of virus matrix proteins, although they show only 2-7% sequence identity to matrix proteins of other virus families (Paramyxoviridae, Rhabdoviridae, Bornaviridae). The late domain sequences important for virus budding are some of the few structurally conserved features present on the proteins of different virus families (Timmins et al. 2004). The crystal structure of a truncated Ebola virus VP40 protein (Dessen et al. 2000) revealed that it consists of two domains with unique folds connected by a flexible linker. At the beginning of the second domain is a trypsin-cleavage site after amino acid 212. The N-terminal cleavage fragment shows spontaneous hexamerization in vitro (Ruigrok et al. 2000). The N-terminal domain of VP40 is involved in the protein-protein interactions required for oligomerization while the C-terminal part is responsible for membrane-binding properties. Denaturing conditions (such as 4M urea) or membrane association induce hexamer formation of full-length VP40 (Scianimanico et al. 2000). VP40-hexamers formed from three antiparallel homodimers line the inside of Filovirus particles. On electron micrographs of Ebola virus VP40 oligomers hexameric and octameric structures were identified (Timmins et al. 2003). The disc-shaped octamers are formed in association with RNA by four antiparallel homodimers of Ebola VP40. The crystal structure revealed that the interaction with RNA induces two conformational changes of the protein (Gomis-Rüth et al. 2003). In contrast to Ebola virus VP40, the Marburg virus matrix protein forms oligomers that stack up to form complex polymeric structures. Therefore the oligomeric status could not be determined by electron microscopy.
In addition to the in vitro characterization of VP40, the cellular mechanisms involved in Filovirus budding from mammalian cells have been studied. Timmins et al. (2001) demonstrated that full-length Ebola VP40, but not the C-terminally truncated protein, is released into the culture supernatant of mammalian cells and that the matrix protein seems to be released inside vesicles. The expression of VP40 alone leads to secretion of few Filovirus-like particles, but co-expression with GP increases the formation of these structures (Noda et al. 2002, Kolesnikova et al. 2004b). Panchal et al. 2003 demonstrated in vivo that oligomers of full length Ebola VP40 are formed only in association with membranes. These oligomers seem to be transported via lipid rafts to the site of assembly. It was also shown that VP40 recruits the cellular protein Tsg101 via specific interaction with an intrinsic late budding domain (PTAP) (Licata et al. 2003). Tsg101 is involved in the vacuolar sorting pathway of mammalian cells and is important for efficient budding of numerous viruses. In addition, hexamers of VP40 can bind to the WW domain of human Nedd4, another protein involved in the late endosomal pathway necessary for efficient virus budding (Timmins et al. 2003). It was demonstrated by Yasuda et al. (2003) that Nedd4 regulates the egress of Ebola virus-like particles from mammalian cells. Despite only 29% sequence homology to the Ebola matrix protein, Marburg virus VP40 protein also associates with membranes of the late endosomal compartment both in virus-infected cells as well as in recombinant expression in mammalian cells (Kolesnikova et al. 2002, Kolesnikova et al. 2004a). While the Tsg101-binding PTAP motif is absent from Marburg virus VP40, the PPXY motif for binding of Nedd4 (Yasuda et al., 2003) is found in Marburg virus and may also to be important for efficient budding. Ebola virus VP40 shows the properties of a microtubule-associate protein (MAP) because of its binding to tubulin. It enhances tubulin polymerization in vitro (Ruthel et al. 2005). It further stabilizes microtubules against depolymerization and could therefore play a role in directed transport of virus particles to the site of budding
Reviews describing the current knowledge of the impact of VP40 on molecular mechanisms involved in Filovirus cellular trafficking (Aman et al 2003) and the current understanding of the process of Filovirus budding (Jasenosky and Kawaoka 2004) have been published.
The nucleoprotein (NP) of Marburg virus self-assembles tubule-like structures when recombinantly expressed in mammalian cells. These aggregates resemble structures observed in sections of viral inclusions of Marburg virus infected cells (Kolesnikova et al. 2000). In association with cellular RNA (when expressed in Sf21 cells), Marburg virus NP forms regularly structured loose coils (Mavrakis et al. 2002). High salt treatment tightens the coiling. Ebola virus NP expressed recombinantly in mammalian cells forms intact nucleocapsids only if expressed in combination with Ebola VP35 and VP24 (Huang et al. 2002). Other virus proteins are not necessary for nucleocapsid formation, but post-translational O-glycosylation of NP is required to facilitate VP35-binding. NP increases the release of Ebola virus-like particles if co-expressed with VP40 in mammalian cells. Further addition of Ebola GP potentiates this effect (Licata et al. 2004).
The structure and function of the minor matrix protein VP24 are not fully understood. While co-expression of VP24 and VP40 in mammalian cells does not increase VLP release, co-expression of VP40, NP and VP24 in mammalian cells yields a higher amount of Ebola VLPs than co-expression of VP40 and NP alone, suggesting that VP24 may interact with NP (Licata et al. 2004). In cells infected with Ebola virus, as well as upon recombinant expression, VP24 appears to be concentrated in the perinuclear region (Han et al. 2003). VP24 from virus infected mammalian cells is not glycosylated via N-linked sugar residues and appears to tetramerize. Studies have further shown that VP24 strongly interacts with lipid bilayers and is potentially located within lipid rafts. Recent studies have shown that VP24 is essential for the formation of a functional ribonucleoprotein complex (Hoenen et al. 2006). In addition, it has been shown that VP24 seems to block proper IFN signaling giving another potential explanation of how Filoviruses evade the host immune responses (Reid et al. 2006).
The two small proteins contained in the nucleocapsid complex have different functions. VP35 is an essential part of the Filovirus nucleocapsid (Huang et al. 2002, Mühlberger et al. 1998) but the exact role in capsid assembly has not been shown yet. VP35 is a type I IFN antagonist and therefore a major contributor to Filovirus pathogenicity (Basler et al. 2003, Hartman et al. 2004). The role of the phosphoprotein VP30 is linked to transcription activation of the nucleocapsid complex. It was shown that phosphorylation on two serine residues (40 and 42) is essential for binding to NP and therefore inclusion into the nuclear core complex (Modrof et al. 2001). Phosphorylation negatively regulates transcription activation mediated by VP30 and phosphorylase inhibition can therefore inhibit Ebola virus growth (Modrof et al. 2002). It was further shown that the effect of VP30 seems to be directed at a very early step in transcription initiation (Weik et al. 2002). VP30 oligomerizes due to an internal cluster of hydrophobic residues (containing four leucine residues). Mutation of the leucine residues or application of a peptide binding to this region abolishes VP30-dependent Ebola virus transcription activation (Hartlieb et al. 2003).
The pathogenesis of Ebola virus infection is still not fully understood, even though progress towards understanding the mechanisms has been made in recent years. One finding with most hemorrhagic fever cases caused by Filoviruses is that lesions caused by the viral infection are not severe enough to account for terminal shock and death of the host. Yet this is the most common cause of death in viral hemorrhagic fevers. This suggests the involvement of inflammatory mediators as an important part in the pathogenic pathways. A simplified primate model (Sullivan et al. 2003b) suggests that initial host immune responses as well as cell damage due to infection of monocytes and macrophages causes the release of pro-inflammatory cytokines. Infection of endothelial cells causes damage to the endothelial barrier which in combination with effects of the cytokines leads to loss in vascular integrity resulting in hemorrhage and vasomotor collapse (primary cause of death in Ebola patients). Another model further elaborates on the detailed chain of events (Geisbert and Jahrling. 2004). The authors suggest that cytokines released by initially infected monocytes and macrophages recruit further macrophages to the sites of infection. This makes more target cells available for viral exploitation and further amplifies the dysregulated host response, leading to loss of lymphocytes by apoptosis. Ebola virus enhances expression of tissue factor, resulting in activation of the clotting pathway and formation of fibrin in the vasculature. These coagulation disorders are complemented by infection of hepatocytes and adrenal cortical cells resulting in impaired synthesis of important clotting factors. Studies of human Ebola Sudan patients showed leucopenia and unresponsive PBMC's, high viral loads in infected monocytes and elevated nitric oxide levels to be associated with a fatal outcome of the disease (Sanchez et al. 2004).
Primates are the only animals known to develop disease following natural infection with Filoviruses. However, in order to research virus biology, several animal models of disease have been developed, including a non-human primate model, guinea pig models, and a mouse model for Ebola virus infection using a mouse-adapted Ebola Zaire virus. Fisher-Hoch and McCormick (1999) describe several of these animal models and a publication by Ryabchikova et al. (1999) reviews animal pathology of Filoviral infections in more detail. Animal models have been used to develop the current pathogenesis model based on in vitro experiments using primate monocyte/macrophage cultures (Feldmann et al. 1996, Ströher et al. 2001) as well as in vivo experiments comparing the coagulation changes in mice, guinea pigs, and monkeys (Bray et al. 2001). While small animal models can provide insight into the virus life cycle and pathogenicity and help as an economical choice for vaccine and antiviral drug development, only non-human primates show a disease progression similar to human disease (Geisbert et al. 2002). Pathogenesis in the cynomolgus macaque model of Ebola Hemorrhagic Fever has been described in detail by Geisbert et al. (2003).
Since there is no treatment to cure Filovirus induced hemorrhagic fevers, the majority of patients succumb to the disease. However, between 10 to 50% (depending on virus strain and medical support available) of infected humans survive the disease. Following the large 1995 outbreak of Ebola Zaire in the Democratic Republic of Congo, sera of patients were analyzed. It was found that while IgM and IgG antibodies appeared at approximately the same time after onset of disease, high IgM titers persisted for a much shorter time in survivors in comparison to fatal cases (Ksiazek et al. 1999). IgG titers in survivors were detectable for at least two years following recovery. Ebola virus patients receiving convalescent sera during their illness have been reported to show a higher survival rate (Mupapa et al. 1999). Based on this observation, immune mouse sera from mice receiving sub-lethal doses of Ebola virus were transferred to naïve immunodeficient mice. Dose-dependent protection against virus challenge was observed in recipients of immune sera (Gupta et al 2001). Recombinant human monoclonal antibodies were constructed via phage display from bone marrow RNA of two Ebola survivors (Maruyama et al. 1999) and in vitro virus neutralization was demonstrated with one of these antibodies (Maruyama et al. 1999b). The same human monoclonal antibody was able to provide pre- and post-exposure prophylaxis in the guinea pig model of Ebola hemorrhagic fever (Parren et al. 2002). Similarly, mouse monoclonal antibodies to Ebola GP applied pre- or post-exposure were successful to protect mice against lethal Ebola virus challenge (Wilson et al. 2000). Another route to provide antibody-based protection against viral diseases is the preparation of hyperimmune serum in non-susceptible animals. Sera prepared in sheep and goats tested successfully for protection in the guinea pig model of Ebola virus (Kudoyarova-Zubavichene et al. 1999). A similarly prepared equine anti-Ebola Zaire serum was tested for protection in baboons, mice and guinea pigs. Results were inconsistent and showed low levels of protection in mice and monkeys, while all guinea pigs were protected against a lethal dose of Ebola virus. While viremia and disease onset in cynomolgus monkeys treated with the equine hyperimmune serum challenged with Ebola virus was delayed, all animals succumbed to the disease when protective antibodies had been depleted (Jahrling et al. 1996, Jahrling et al. 1999). One potential reason for the failure of passive immunizations in non-human primates could be antibody-dependent enhancement of infection (as demonstrated in vitro by Takada et al. 2003b). A further hurdle for the prophylactic or therapeutic use of neutralizing antibodies could be strain specificity. Out of five different neutralizing epitopes that have been identified on Ebola Zaire GP, none seems to have neutralizing activity against glycoproteins from the Sudan, Ivory Coast or Reston strains of Ebola virus or against the glycoprotein of Marburg virus (Takada et al. 2003).
In contrast to the mostly dramatic symptomatic cases of Filovirus infections, asymptomatic cases have been reported (Leroy et al. 2000). In only a few of these cases, specific antibody responses against Ebola virus proteins could be detected. Interestingly, the responses were directed against NP as well as VP40, and not against Filovirus glycoprotein, which is thought to be the main protective antigen and the target for virus neutralizing antibodies. The asymptomatic individuals furthermore showed early and strong inflammatory responses with high levels of circulating cytokines. These clinical parameters emphasize the importance of proper humoral and cell-mediated immunity in protection against Filovirus infection. Induction of cytotoxic T cells directed against GP was observed upon immunization of mice with liposome-encapsulated irradiated Ebola virus particles (Rao et al. 1999). Responses seem to be dependent on proper delivery of antigen to antigen presenting cells (APC). It was further demonstrated that the liposome-encapsulated virus particles can induce T cell help necessary to protect mice against lethal challenge (Rao et al. 2002). However, the same vaccine did not provide good protection in a primate model. Another study reports the generation of protective cytotoxic T lymphocytes after 2 or 3 immunizations with recombinant VEE replicons expressing Ebola NP (Wilson and Hart 2001). Even though the cellular response achieved did not yield 100% protection in the mouse model, it provides evidence that proper cellular responses will aid in protection. Identification of CTL epitopes on the nucleoprotein may lead to the discovery of potential defense mechanisms (Simmons et al. 2004). Another experiment in the mouse model demonstrated that cytotoxic memory T cells alone can protect against death, but that a persistent infection can develop if mice are deficient in helper T-cells or mount an insufficient humoral response (Gupta et al. 2004). This suggests a potential mechanism for development of a natural reservoir host animal for Filoviruses.
One of the characteristic features of Filovirus disease in infected primates is the rapid decline in the number of circulating lymphocytes. It was shown that Ebola virus induces apoptosis in natural killer cell populations by day two post-infection (Reed et al. 2004). It may therefore inhibit activation of lymphocytes by eliminating the subsets that are most likely to be capable of mounting an effective response to the virus. The viral component responsible for this has not been identified yet, although Ebola virus immune suppression seems to be linked to a domain of the surface glycoprotein and/or the VP35 protein. Studies with Ebola and Marburg virus-like particles containing GP and VP40 (Bosio et al. 2004) demonstrated activation of human myeloid dendritic cells. In contrast, administration of live or inactivated Ebola virus does not induce dendritic cell maturation. Other virus proteins not contained in the recombinant particles may be responsible for preventing this early immune response. Particles containing only the viral matrix protein VP40 were able to specifically activate natural killer cells and induce an early protective immunity against Ebola virus infection 1-3 days after immunization (Warfield et al. 2004). It was shown that the protection was mediated by perforin-mediated lysis of target cells by NK cells. This highlights another pathway that may be critical to provide successful protection against Filovirus infection.
Conventional vaccine approaches against Filovirus infection, such as attenuated or killed Filovirus particles are not generally considered feasible due to the high safety risks involved. Demonstration of proper attenuation of live viral strains in humans would be much too risky to consider and the risk of reversion would further complicate this approach. For example, a serendipitously acquired virus strain which had proven to be nonlethal to strain 13 guinea pigs proved to be lethal to Hartley guinea pigs (Hevey et al. 2002). Production of large quantities of virus to generate a killed vaccine would likewise be unacceptable from a safety point of view. Instead, several modem vaccine approaches have been the major area of research. Approaches evaluated or under evaluation include DNA vaccines, adeno-, alpha- or vesicular stomatitis virus vectored vaccines, and recombinantly produced proteins. Summarizing overviews of the advances in developing vaccines to protect against Filovirus induced hemorrhagic fevers were published in several review papers (Enserink 2003, Hart 2003, Geisbert and Jahrling 2004).
Xu et al. (1998) reported successful protection of guinea pigs against Ebola virus infection after injecting animals with 3 doses of DNA vaccines expressing either Ebola surface glycoprotein, the soluble glycoprotein sGP, or the nucleoprotein. A similar approach (Vanderzanden et al. 1998) reported successful protection of mice against Ebola virus infection after gene gun application of 4 or 5 doses of DNA vaccines encoding Ebola GP or NP. Protection of non-human primates against Ebola virus infection has been demonstrated after immunization with three doses of a combination of DNA vaccines followed by a boost utilizing a recombinant adenovirus vector expressing Ebola Zaire GP (Sullivan et al. 2000). The Vaccine Research Center at NIH is currently developing a prime-boost vaccine for human use based on this approach. It has further been shown that a single dose of the recombinant adenovirus can elicit a protective response against Ebola virus challenge four weeks after immunization (Sullivan et al. 2003). Data provided by these immunological studies strongly suggest the importance of Ebola GP in development and persistence of vaccine protection (Sullivan et al. 2003b). DNA vaccines need to be applied many times in order to raise effective immune responses. They could potentially lead to the development of autoimmune diseases and the use of strong promoters contained in the used plasmids may present a safety risk in human application. Recombinant adenoviruses may not be efficacious as vaccines on individuals with pre-existing immunity to adenoviruses and can for the same reason only be applied once. Due to safety concerns, development of adenoviral vectors for gene therapy and vaccine development has slowed in past years. In a clinical trial using adenovirus vectors for gene therapy, a patient died due to complications associated with the use of Ad5 virus (Raper et al. 2003).
Another approach for vaccine development focused on the use of alphavirus (VEE) replicons. Using vectors expressing several Marburg virus structural proteins, Hevey et al. (1998) were able to demonstrate that guinea pigs developed high specific serum antibody titers and could be protected if Marburg GP, NP or VP35 proteins were applied. Marburg virus VP40, VP30 or VP24 did not protect against viral challenge. Analogous vectors expressing Ebola virus GP, NP, VP24, VP30 or VP40 (Wilson et al 2001, Pushko et al. 2001) showed a significant level of protection against homologous challenge in vaccinated mice and guinea pigs. Passive transfer of immune sera was unsuccessful in providing protection against disease.
Recently a new vaccine approach has been tested utilizing replication-competent vesicular stomatitis virus vectors expressing surface and secreted glycoproteins of Ebola Zaire and Marburg viruses. A VSV vector expressing Ebola surface GP protected mice against lethal challenge (Garbutt et al. 2004). Further studies showed protection against homologous (but not heterologous) Filovirus challenge in guinea pigs and cynomolgus monkeys (Jones et al. 2005). Daddario-DiCaprio et al. (2006) showed that this vaccine may even provide post-exposure protection if administered at a high dose within 20-30 minutes after infection.
Virus-like particles based on recombinant proteins expressed in mammalian cell lines have been evaluated for their potential as protective vaccines against Filovirus infections. Particles containing Ebola GP and VP40 have shown to be more successful in protecting mice against lethal Ebola virus challenge than inactivated virus particles (Warfield et al. 2003). This indicates that virus inactivation may destroy necessary native structural features on the contained virus proteins. It was further demonstrated that Marburg virus-like particles constructed using Marburg virus GP and VP40 successfully induced protection against Marburg virus infection in guinea pigs, while Ebola VLPs were not protective against Marburg virus challenge (Warfield et al. 2004). Only a vaccine containing both Marburg and Ebola virus-like particles was able elicit protection against challenge with both types of Filoviruses in the guinea pig model (Swenson et al. 2005) demonstrating a lack of cross-protection between Filoviruses.
Marburg virus GP was expressed by baculovirus recombinants in Sf9 and Trichoplusia ni cells as full-length or truncated version without the transmembrane region. Culture supernatants formulated with Ribi adjuvant (Jennings, V. M. Review of Selected Adjuvants Used in Antibody Production. ILARJ 37:119-125. 1995) were utilized to immunize guinea pigs. Protection was achieved only against the homologous Marburg virus strain (Hevey et al. 1997). It was furthermore demonstrated that passive transfer of immune sera conferred protection to naïve guinea pigs. Baculovirus expressed full length or truncated Ebola GP was administered to guinea pigs alone (2 doses) or in a prime-boost scheme following one dose of a DNA vaccine. Prime-boost and the protein-only vaccines (applied with adjuvant) induced protective immunity in less than 50% of the animals (Mellquist-Riemenschneider et al. 2003).
An article published by Hevey et al. 2002 compared the protection in guinea pigs provided by several Marburg virus GP-based vaccine strategies. Approaches included a DNA vaccine, VEE-replicons, baculovirus expressed recombinant GP or a prime-boost approach combining DNA vaccine and recombinant protein. The study suggested that DNA vaccines and replicon-based vaccines were particularly interesting. Hart (2003) cautions that it may be dangerous to focus a vaccine approach solely on the surface glycoproteins. Certain individuals may not develop the desired T-cell responses to GP and may therefore not be protected.
Adjuvants are materials that increase the immune response to a given antigen. Since the first report of such an enhanced immunogenic effect by materials added to an antigen (Ramon, G., Bull. Soc. Centr. Med. Vet. (1925) 101:227-234), a large number of adjuvants have been developed, but only calcium and aluminum salts are currently licensed in the United States for use in human vaccine products. Numerous studies have demonstrated that other adjuvants are significantly more efficacious for inducing both humoral and cellular immune responses. However, most of these have significant toxicities or side-effects which make them unacceptable for human and veterinary vaccines. In fact, even aluminum hydroxide has recently been associated with the development of injection site granulomas in animals, raising safety concerns about its use. Because of these problems, significant efforts have been invested in developing highly potent, but relatively non-toxic adjuvants. A number of such adjuvant formulations have been developed and show significant promise (Cox, J. C. and Coulter, A. R., Vaccine (1997) 15:248-256; Gupta, R. K. and Siber, G. R., Vaccine (1995) 13:1263-1276), especially in combination with recombinant products. Several of these modern adjuvants are being tested in preclinical and clinical trials designed to examine both efficacy and safety.
The main modes of action of adjuvants include (i) a depot effect, (ii) direct immunomodulation through interaction with receptors, etc., on the surface of immune cells and (iii) targeting antigens for delivery into specific antigen-presenting cell populations (e.g., through the formation of liposomes or virosomes). The depot effect results from either the adsorption of protein antigens onto aluminum gels or the emulsification of aqueous antigens in emulsions. In either case this results in the subsequent slow release of these antigens into the circulation from local sites of deposition. This prevents the rapid loss of most of the antigen that would occur by passage of the circulating antigen through the liver. Immunomodulation involves stimulation of the “innate” immune system through interaction of particular adjuvants with cells such as monocytes/macrophages or natural killer (NK) cells. These cells become activated and elaborate proinflammatory cytokines such as TNF-α and IFN-γ, which in turn stimulate T lymphocytes and activate the “adaptive” immune system. Bacterial cell products, such as lipopolysaccharides, cell wall derived material, DNA, or oligonucleotides often function in this manner (Krieg, A. M. et al., Nature (1995) 374:546; Ballas, Z, J, et al., J. of Immunology (2001) 167:4878-4886; Chu, R. S., et al., J. Exp. Med. (1997) 186:1623; Hartmann, G. and Krieg, A., J. Immunol. (2000) 164:944-952; Hartmann, G., et al., J. of Immunol. (2000) 164:1617-1624; Weeratna, R. D. et al., Vaccine (2000) 18:1755-1762; U.S. Pat. Nos. 5,663,153; 5,723,335; 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; 6,429,199). Targeting of antigens to (and within) antigen presenting cells is accomplished through the delivery and fusion of antigen bearing vehicles (e.g., liposomes or virosomes) with antigen presenting cells, thereby delivering the antigen into the intracellular pathways necessary for presentation of antigen in the context of MHC Class I and/or II molecules (Leserman, L., J. Liposome Res. (2004) 14:175-89; Bungener et al., Vaccine (2005) 23:1232-41).
In addition to these more traditional adjuvants, various new technologies are under development. One of them includes the use of dipeptidyl peptidases, which have shown promising results in human cancer therapy. The biological activity of dipeptidyl peptidases may reflect adjuvant-like properties (e.g. generating cytokine and chemokine production), as shown in preclinical animal models in response to their administration. These responses are believed to enhance both antigen-presentation to naive T-cells and the co-stimulation of antigen-specific T-cells. The use of adjuvants in combination with recombinant antigens is widely known in the art and can result in vaccines with better efficacy.
Thus, there is an unmet need for vaccines against Filoviruses. A key technical problem to be solved is the efficient production of conformationally relevant Filovirus surface glycoproteins, as well as additional structural proteins, that serve as potent immunogens in vaccinated subjects. Related technical problems are the production and formulation of effective vaccines that have improved costs of production and distribution.